It is fairly well established that many pathological conditions, such as infections, cancer, autoimmune disorders, etc., are characterized by the inappropriate expression of certain molecules. These molecules thus serve as “markers” for a particular pathological or abnormal condition. Apart from their use as diagnostic “targets”, i.e., materials to be identified to diagnose these abnormal conditions, the molecules serve as reagents which can be used to generate diagnostic and/or therapeutic agents. A by no means limiting example is the use of a peptide which complexes with an MHC molecule, to generate cytolytic T cells against abnormal cells.
Preparation of such materials, of course, presupposes a source of the reagents used to generate these. Purification from cells is one laborious, far from sure method of doing so. Another preferred method is the isolation of nucleic acid molecule which encode a particular marker, followed by the use of the isolated encoding molecule to express the desired molecule.
To date, two strategists have been employed for the detection of such antigens, in e.g., human tumors. These will be referred to as the genetic approach and the biochemical approach. The genetic approach is exemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85:2275 (1988), incorporated by reference. In this approach, several hundred pools of plasmids of a cDNA library obtained from a tumor are transfected into recipient cells, such as COS cells, or into antigen-negative variants of tumor cell lines which are tested for the expression of the specific antigen. The biochemical approach, exemplified by, e.g., O. Mandelboim, et al., Nature 369:69 (1994), incorporated by reference, is based on acidic elution of peptides which have bound to MHC-Class I molecules of tumor cells, followed by reversed-phase high performance liquid chromography (HPLC). Antigenic peptides are identified after they bind to empty MHC-Class I molecules of mutant cell lines, defective in antigen processing, and induce specific reactions with cytotoxic T-lymphocytes. These reactions include induction of CTL proliferation, TNF release, and lysis of target cells, measurable in an MTT assay, or a 51Cr release assay.
These two approaches to the molecular definition of antigens have the following disadvantages: first, they are enormously cumbersome, time-consuming and expensive; and second, they depend on the establishment of cytotoxic T cell lines (CTLs) with predefined specificity.
The problems inherent to the two known approaches for the identification and molecular definition of antigens is best demonstrated by the fact that both methods have, so far, succeeded in defining only very few new antigens in human tumors. See, e.g., van der Bruggen, et al., Science 254:1643-1647 (1991); Brichard, et al., J. Exp. Med. 178:489-495 (1993); Coulie, et al., J. Exp. Med. 180:35-42 (1994); Kawakami, et al., Proc. Natl. Acad. Sci. USA 91:3515-3519 (1994).
Further, the methodologies described rely on the availability of established, permanent cell lines of the cancer type under construction. It is very difficult to establish cell lines from certain cancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg. Clin. North. Am. 10:607-637 (1990). It is also known that some epithelial cell type cancers are poorly susceptible to CTLs in vitro, precluding routine analysis. These problems have stimulated the art to develop additional methodologies for identifying cancer associated antigens.
One key methodology is described by Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11913 (1995), incorporated by reference. Also, see U.S. Pat. No. 5,698,396, incorporated by reference. To summarize, the method involves the expression of cDNA libraries in a prokaryotic host. (The libraries are secured from a tumor sample). The expressed libraries are then immunoscreened with absorbed and diluted sera, in order to detect those antigens which elicit high titer humoral response. This methodology is known as the SEREX method (“Serological identification of antigens by Recombinant Expression Cloning”). The methodology has been employed to confirm expression of previously identified tumor associated antigens, as well as to detect new ones. See the above referenced patent applications and Sahin, et al., supra, as well as Crew, et al., EMBO J. 144:2333-2340 (1995).
One important antigen identified by the SEREX methodology is referred to as NY-ESO-1. The antigen is described in, e.g., U.S. Pat. No. 5,804,381, and Chen, et al., Proc. Natl. Acad. Sci. USA 94:1914-1918 (1997), the disclosures of which are incorporated by reference. Originally, NY-ESO-1 was characterized as an antigen which was processed into peptides presented by MHC Class I molecules. Later work showed that it also processed into peptides that are presented by Class II molecules. See Jäger, et al., J. Exp. Med. 191:625 (2000), as well as PCT application publication number WO99/53938, published Oct. 28, 1999, both of which are incorporated by reference in their entirety. Also, see WO 01/23560, also incorporated by reference. Additional papers have been published which describe additional peptides which consist of amino acid sequences found in NY-ESO-1, which also bind to MHC Class II molecules and serve as T cell epitopes. Exemplary are Zeng, et al., J. Immunol. 165:1153-1159 (2000); Zarour, et al., Canc. Res. 60:4946-4952 (2000); Zarour, et al., Canc. Res. 62:213-218 (2002); Zeng, et al., Proc. Natl. Acad. Sci. USA 98(7):3964-3969 (2001), and Zeng, et al., Canc. Res. 62:3630-3635 (2002), all of which are incorporated by reference.
The interest in such molecules results from several factors. First, NY-ESO-1 appears to be restricted in its expression to tumor cells, of various histological types, and male germ cell lines. Exemplary of the tumor types in which NY-ESO-1 expression is found are melanoma, breast, prostate, lung, urinary bladder, carcinoma, and synovial sarcoma. See Jäger, et al., supra. Also see Chen, et al., supra, Stockert, et al., J. Exp. Med. 187:1349 (1998); Wang, et al J. Immunol. 161:3598-3606 (1998); Jungbluth, et al. Int. J. Cancer 92:856-860 (2001); Jungbluth, et al, Int. J. Cancer 94:252-256 (2001); all incorporated by reference.
The fact that T cells play an important role in controlling tumor growth and mediating tumor regression is well known. The molecular mechanisms underlying T cell mediated anti-tumor immunity has been elucidated, inter alia by the identification of tumor antigens that are recognized by CD8+ T cells. See Rosenberg, Immunity 10:281-287 (1998); Wang, et al., Immunol. Rev. 170:85-100 (1999). The advances in the identification of such molecules have led to their use in clinical trials, examples of which may be seen in Rosenberg, Nature 411:380-384 (2001). Also see Nestle, et al., Nat. Med. 4:328-332 (1998); Rosenberg, et al., Nat. Med. 4:321-327 (1998); Lee, et al., J. Clin. Oncol. 19:3836-3847 (2001); Thurner, et al., J. Exp. Med. 190:1669-1678 (1999).
The growing interest in Class II presentation stems, in part, from animal model studies that indicate that it may be necessary to engage CD4+ cells as well as CD8+ cells in order to develop effective cancer vaccines. See Zeng, J. Immunother 24:195-204 (2001).
To move from the general back to the specific, NY-ESO-1, as has been pointed out, supra, shows strict tumor expression. In addition to the CD8+ response noted supra, high titers of NY-ESO-1 antibodies were present in patients who express the molecule, suggesting that there is a CD4+ response involved. See Wang, et al, Immunol. Rev. 179:85-100 (1999); Pardoll, et al., Curr. Opin. Immunol 10:588-594 (1998); and Jager et al., Proc. Natl. Acad. Sci. USA 97:4760-4765 (2000). While NY-ESO-1 derived peptides (“derived” as used herein, refers to amino acid sequences which can be found in the NY-ESO-1 protein sequence described in the Chen '381 patent and PNAS paper cited supra) have been identified which are presented by HLA-DRB1*0401 and HLA-DRB1*0101 (Zeng, et al., J. Immunol. 165:1153-1159 (2000), Jäger, et al., J. Exp. Med. 191:625-630 (2000)), the majority of patients who present NY-ESO-1 specific antibodies do not present these MHC-Class II molecules. Hence, there is an interest in finding additional peptides, derived from NY-ESO-1, which bind to MHC-Class II molecules, for all of the reasons described supra. Further, there is a need to extend the use of peptide vaccines to patients who do not present MHC-Class II molecules.
There are various “rules” or “guidelines” for determining if a peptide of interest should bind to a given MHC or HLA molecule. See, for example, Marsh, et al., The HLA Factsbook (Academic Press, 2000), which presents a listing of “binding motifs” for determining the likelihood of a peptide binding to a particular MHC Class I or Class II molecule. There are also numerous algorithms and programs available to facilitate this review. See, e.g., Southwood, et al., J. Immunol. 160:3363 (1998); Honeyman, et al., Nat. Biotechnol. 16:966-969 (1998); Breisie, et al., Bioinformatics 14:121-131 (1998), as well as the “SYFPEITHI” algorithm, referred to infra. As will be shown, experimental conformation of these algorithms is always required before any conclusions can be drawn. The common occurrence of false positives is a major drawback of algorithm defined, HLA binding peptides.
The disclosure which follows identifies a new, promiscuous Class II binding peptide, derived from NY-ESO-1. The ramifications of this discovery are also a part of this invention, as will be seen from the disclosure which follows.